M.' LURDES SADLER SIMÕES
M.' CÃNDIDAT. ABREU VAZ
and J. J. R. FRAÚSTO da SILVA
Centro de Química Estrutural
Complexo Interdisciplinar
1ST — 1000 Lisboa — Portugal
1 — INTRODUCTION
It is well known that some plants like spinach,
lettuce or beet are generally rich in nitrate; this level
becomes higher if nitrate fertilizers are used. As nitrate may be reduced to nitrite in fresh products
after harvest, the nitrite being toxic in low levels,
the interest of an accurate determination of these
species has increased in the last decade.
The primary toxicity of nitrite is manifested
through oxidation of iron in hemoglobin from the
ferrous to the ferric form, and there have been reports of infants with ages between two and twelve
months experimenting more or less severe symptoms of met haemoglobinaemia after eating spinach
[1, 2]. Furthermore, in acid medium, nitrite can
react with amines of nucleic acids to form nitrosamines which can originate alterations in the genetic
code. This type of reaction is general: N-nitroso
compounds are formed by reaction of nitrite with
secondary amines [l,3,13].Taking into account the
SELECTION OF
ANALYTICAL METHODS
FOR THE
DETERMINATION OF
NITRATE AND NITRITE
IN VEGETABLES f''
great variety of amines of this type existing in food
and as the rate of formation of N-nitroso compounds is proportional to the square of nitrite concentration, efforts should be made to reduce the
amount of nitrite ingested. This is a difficult task
because it is usual to fertilize in excess to increase
the yield and quality of crops. Moreover, in spite of
its disadvantages and since there is no substitute,
the nitrite ion is used as a preservative and
bactericide in smoked meat to prevent the proliferation of Closiridrtm botulittiunt and improve its colour and taste.
The World Health Organization has established
provisional limits to the levels of nitrate and nitrite
in some kinds of food — 500 mg/kg for nitrate (as
NaNO,) and 200 mg/kg for nitrite (as NaNO,) for
canned and smoked meat [42]. This Organization
has also considered that nitrate and nitrite ingested
by man should not exceed a maximum daily limit of
5 mg/kg and 0.2 mg/kg body weight [42].
Owing to the necessity of respecting such limits and
since there are no official methods for nitrate and
Among the analytical methods for determination of nitrate and
nitrite determination in plants, the aim of this work
nitrite in vegetables three were chosen and studied in terms of
is to select, among the methods reported in the lite-
accuracy, precision, sensitivity, detection limit, ease of performance, safety and practical convenience for routine use. Some
rature, those which can be easily used in control and
suitable modifications have been introduced and the two me-
research laboratories without auto-analysers.
thods selected for nitrate and nitrite determination are described
(17 Part of this work has been presented at the «I.° Encontro
in detail.
Nacional dc Quimica» — Lisboa — Portugal (1978).
Rev. Port. Quím., 21, 61(1979)
61
M. L. SADLER, M. C. T. ABREU VAZ, J. J. R. FRAÚSTO DA SILVA
Considering the importance of spinach in children's
diet this was the vegetable chosen for our study
although the conclusions can be easily extended to
any other type of plants.
Different methods for nitrate determination can be
found in the literature; such methods, mainly
applied to vegetables, include polarography [4-6],
potentiometry with nitrate selective electrode using
different modes of extraction [7-11], spectrophotometry in the ultraviolet range [12, 28] applicable to
the determination of nitrate and nitrite and spectrophotometry in visible range using the strong colour
of the azo compound formed by reaction of the
nitrite with aromatic amines [14, 20-24, 29]. Nitrate
ion is indirectly determined using the same methods
after having been quantitatively reduced to nitrite
using a reducing cadmium column [36, 37]. Considering the carcinogenic character of the aromatic
amines some authors [38] use amines with sulfonic
groups to reduce this inconvenience [39-41], an important improvement for routine methods.
The spectrophotometric methods applicable only to
the nitrate ion should also be mentioned; their
application is based on the reaction of this ion
with diphenylamine [25] and diphenylamine and
p-diaminodiphenylsulphone [35], with Re(V) and
-furildioxime [27, 33], with salicylic acid in alkaline medium [15, 16], with phenazone [17, 29],
with 2,4-xylenol [18], with 2,6-xylenol [19, 26], with
brucine [32] or phenoldisulfonic acid [34].
Another method to be considered is the steam destillation method [30] for determination of ammonium, nitrate and nitrite.
Attending to their accuracy, precision, sensitivity,
detection limit, ease of performance, safety and
practical convenience for routine use, the following
three methods were selected and tried in laboratory
after suitable modifications had been introduced.
1 — Nitrate selective electrode potentiometry
2 — Cleve's Acid reagent spectrophotometry
3 — Steam distillation
After experimental study of these three methods,
the first has been selected for determination of nitrate and the second for determination of nitrite.
Each of these methods is described in detail
including the modifications introduced by us on
account of the great interest of this subject and in
order to make known the techniques extending
them to the analytical chemistry control laboratories.
62
2 — DETERMINATION OF NITRATE
IN SPINACH
(Selective electrode potentiometry)
We have adopted the method described by Mn u - ier
ai. [10); its main advantages compared with the non
automatic colorimetric methods are the shorter time
of analysis and an easier preparation of standards
and samples.
The extracting solution for samples has also
complexation and precipitating effects, thus making
it possible to eliminate most interferences 1101.
2.1 — EXPERIMENTAL
2.1.1. — EQUIPMENT AND REAGENTS
— Orion Nitrate «Specific» electrode, Model 92-07
—
—
—
—
—
—
(Orion Research Incorporated, Cambridge Massachusetts, U.S.A.).
Reference Electrode (SCE), Corning n.° Cat.
476109.
Coleman pH meter (model 38A) with an accuracy of ± 0.1 mV.
Salt bridge saturated in potassium sulphate and
6% in agar-agar.
Kenwood mixer.
Standard solution 1000 ppm in nitrate — prepared with potassium nitrate p.a.
Extracting solution — 0.010 M in aluminium sulphate, 0.010 M in silver sulphate, 0.020 M in boric
acid and 0.020 M in sulphamic acid, adjusted
to pH =3.0 with 0.1 M potassium hydroxide.
2.1.2 — PROCEDURE
CALIBRATION CURVE
In order to establish the calibration curve,
standards were prepared containing between 10 and
1000 ppm of NOS diluted 1:1 with extracting solution. It was found that the slope of the straight line
E = f (log c) was reproducible with time, although
its value was higher than that predicted by Nerst
equation.
All the readings were done by shaking the solution
with a magnetic stirrer during 3 minutes and reading
the value 2 minutes after stirring had stopped.
Rev. Pun. Quím., 21, 61(1979)
DETERMINATION OF NITRATE AND NITRITE
— Kenwood mixer.
PREPARATION OF THE SAMPLES
— Electromagnetic stirrer.
Fresh leaves of spinach were washed, air dried and
— Buffer solution pH = 9.6 — 9.7 (0.67 M in
ground. Samples of 5 to 10 g were weighed and ni-
NH,CI and NH,).
trate was then extracted using 100 ml of extracting
— Diluted buffer solution 1:10 in volume).
solution and 50 ml of water. After 1 hour stirring,
— Sodium hydroxide solution 2.5 Ni.
— 1,7 Cleve's acid solution — 60 mg of Cleve's
the volume of the solution was made up to 200 ml.
The sample was filtered and the nitrate level
acid (BDH) were dissolved in 50 ml of hot distilled
determined by direct pot ent iomet ry.
water. After cooling, 3-4 g of powdered zinc were
added to the light red solution and this was stirred
Notes
for 2-3 minutes. After filtering into a brown bottle,
It should be pointed out that relatively to Mn \.I et
was shaken and stored in a cool place.
50 ml of acetic (99-100%) were added. The solution
a/. 's paper ]10].the following changes have been introduced:
Sulphanilic acid solution — 600 mg of sulphanilic acid were dissolved in 50 iii of hot distilled
1 — The reference electrode Radiometer 601 with
saturated solution of sodium sulphate used by the
authors has not been used. A saturated calomel
electrode and a salt bridge of saturated potassium
sulphate and 6% in agar-agar were preferred instead; this proves to be more economical. Saturated
sodium sulphate solutions made 6% in agar-agar do
water. 50 ml of acetic acid (99-100%) were added
and the solution was shaken and stored in a brown
bottle.
— Fundamental solution for _II \ors met hod 1101.
This solution was prepared mixing equal parts of
the above referred Cleve's acid solution and sulphanitic acid solution.
— Extracting solution:
not solidify at room temperature.
2 — The recommended four minute extraction of
100 mg of ground plant with 5 ml of extracting solution and 5 ml of water has not been followed as
100 mg was considered to be an insufficient sample;
I hour extraction was adopted although a 30 minute
period would be enough. In the first case a more
easily filtered solution is obtained and the time of
the experiment is not remarkably increased since it
is possible to extract the samples while the standards
50 g of CdCI,. 2 1/2 H,0 and 50 g of BaC1,. 2 1/2
H,0 were dissolved in about 1 liter of distilled water.
pH was adjusted to 1 with NCI and diluted to 1 liter
with distilled water.
— Nitrite stock solution — 0.1500 g of sodium
nitrite were dissolved in distilled water in a volumetric flask (1000 mI). Add 5 ml of buffer solution
and make up to volume.
— Activatet charcoal — Darco G 60 — Fluka.
Other types of charcoal did not give reproducible
are being prepared
results.
3 — DETERMINATION OF NITRITE
3.1.2. — PROCEDURE
IN SPINACH
(Cleve's acid reagent spectrophotometry)
The spectrophotometric method used was the one
described by AuRI s.si and Roads'
k•
1381 which in-
volves the use of a non-carcinogenic product .
CALIBRATION CURVE
20 ml of nitrite stock solution were transferred into
a 500 ml volumetric flask, 5 ml of buffer solution
were added and the solution was made up to volume. 0.0, 5.0, 10.0, 15.0, 25.0, 50.0 and 75.0 nil
3.1
—
EXPERIMENTAL
aliquots were transferred into 200 ml volumetric
flasks, !0 ml of buffer solution were added and the
3.1.1. — EQUIPMENT AND REAGENTS
solution was made up to volume. 10 ml of this
solution were transferred to a 25 ml volumetric
— Visible and ultraviolet spectrophotometer, Hita-
flask, 10 nil of the «fundamental solution» were
chi-Perkin-Elmer, model 139, using 1 cm cells.
added and diluted to volume with distilled water.
Rev. Port. Quím., 21, 61(1979)
63
M. L. SADLER SIMÕES, M. C. T. ABREU VAZ, J. J. R. FRAÚSTO DA SILVA
The flasks were placed in a water bath at 25-30° for
(NH,/NH 4 CI) were transferred to a 50 nil volume-
30 minutes and t he absorvance at 530 nm was mea-
tric flask, diluted to volume with clear filtrated and
mixed. I5 nil of this solution were transferred to a
sured with a spectrophotometer using 1 cm cells.
25 ml volumetric flask, 10 ml of the method's ufunSAMPLE PREPARATION
damental solution» were added and the nitrite
Following the method proposed by SCHni i and H si•
content determined. .Although the extracting solu-
[20], 20 g of spinach previously washed, air
tion used is acid the nitrite ion is not lost according
CnFR
metric flask; 100 nil of extracting solution, I g of
to the reaction 3HNO. F H +NO;+2NO + H2O
because the nitrate level is Winch higher than the
Fluka activated charcoal (Darco G-60) and 50 ml of
nit rite one.
dried and ground were transferred to a 200 nil volu-
Notes
distilled water were added. The flask was shaken
mechanically for 1 hour and then 16 ml of sodium
1 — Sc n \t and H v,c urk [20] extraction method was
hydroxide (2.5 si) were added and diluted to volume
preferred to that indicated by
with distilled water. The solution was iiiiniediately
[38], because it is not necessary to add the clarifying
AoRt \ \NS1-
and
Rot;itt Rs
filtered, first through a fast filter paper and then
solution of zinc sulphate and potassium ferrocya-
through a slow filter paper. 5 ml of buffer solution
nide, thus simplifying operations.
Table I
Results for standard solutions
Sensitivity
Mean
Detection limit
(slope of
Analytical
Deviation
(Ng/I) in
calibration
met hod
of standards
nitrogen
Correlation
straight line)
coefficient
+
— 6-5 .3 — 0.5 niV
0.999
Selective
electrode
potent iome-
2.0 m V
14.0
try (NO -1 )
Cleve's
reagent
spectrophotometry (NO-)
Cleve's
reagent
spectrophotometry (NOT)
0.005
absorvance
3.0
0.9 ppm -1
1.000
3.0
0.9 ppm t
0.999
units
0.010
absorbance
hi
units
Steam
Distil-
175
lation
a) The slope of the straight line E = f (log [NO1) is — 65.3 ± 0.5, a value which is somewhat higher than that predicted by Nernst
equation; it is, nevertheless fairly reproducible.
b) The highest value of the mean deviation for nitrate standards is due to the errors introduced by the reduction of nitrate to nitrite in the
cadmium column.
64
Rev. Port. Quím., 21, 61(1979)
DETERMINATION OF NITRATE AND NITRITE
2 — The addition of activated charcoal to the
4.1 —
standards according to ADRIAANSE and ROBBERS [38]
STUDIES WITH STANDARD
SOLUTIONS
was unnecessary and therefore omitted.
The selected methods have been studied in terms of
This method can also be used for nitrate determina-
reproducibility of standards, detection limits, sensi-
tion after reducing it to nitrite in a cadmium co-
tivities and correlation coefficient of the straight
lumn. However the process is delayed by this opera-
lines (Table I).
tion because reproducible results can only be
STUDIES WITH SPINACH SAMPLES
obtained if the flow of the solution through the
4.2 —
column is rigorously controlled. This flow must be
4.2.1 — RECOVERY OF ADDED AMOUNTS
controlled to obtain the maximum value of absor-
OF STANDARD
vance, which means a quantitative reduction of niAfter having studied the best experimental condi-
trate ion to nit rite wit how reducing t he latter.
The BREMNER and ht t Nrv's [30] steam distillation method which has also been studied experimentally
and in which some alterations were introduced is
tions, the recovery percentage of an amount added
to the sample was determined.
4.2.2 — REPRODUCIBILITY OF THE METHODS
not described in detail. The most important alteration consists in changing the extracting solution:
instead of 1 M potassium chloride, sett si t and
HATR,w - R [20]
extracting solution was used and so this
method became more reproducible and accurate.
In order to estimate the reproducibility of each one
of the methods, determinations were made for sets
of ten aliquots of the same previously homogenized
sample, except for the nitrite ion where four determinations were made. In this case the scattering of
the results is greater because the values for fresh
4 — RESULTS AND DISCUSSION
spinach are very close to the detection limit of the
In order to determine the best conditions to apply
method.
Table III
the different methods, Iheir accuracy, sensitivity
Reproducibility of the analytical methods studied
and precision, an experimental study has been
carried out on a comparative basis.
\nal\tical
met hod
Table II
elect rode
potentiome_
try (NM)
by the described methods
method
Added concentraDetermined
lion (nitrogen ppm) concentration
% Recovery
Selective
62.4
66.0
potentio-
82.1
76.0
metry (NÕ1 )
143.4
151.0
Qn
error
reagent
105.0 "'
92.6
tometry (NÕ,)
105.3
Cleve's
reagent
spectropho-
Cleve's
,
10--5x10-
0.7x10 '
ca.
10 ` — 4 x 10'
0.25 x 10 -5
ca. loco
,
4
10` — 5 x 10
5 x 10 4
ca. 6%
,
t0`
1x10
I°'a
Cleve's
sped ropho_
electrode
Relatoc
range
(in nitrogen)
Sclect is e
Recovery percentage of an added sample analysed
Analytical
Percentage
tometry (NÕ; )
Reagent
spectrophotomerry (NO 2 )
0.85
0.83
97.6
Steam distillation(NO7)
-
5x10
2
1
ca. 2°o
Cleve's
reagent
0.64
spectrophotomnetry (NO7)
0.63
98.6
a) It should be pointed out that in the direct potentiometry an
error of ± I mV in the junction potential between the salt
bridge and the sample and between the same salt bridge and
Steam distillation
13.37
13.30
99.5
NOT + NO; 21.49
NHt+ NO 28.42
21.70
100.9
29.05
102.2
NH 4 *
Rev. Port. Quím., 21, 61(1979)
each one of the standards leads to an error of ± 4%. So the
results may be considered to be within the expected values.
It should also be noted that a method which is not accurate
i
may have good recoveries of an added amount of a standard,
the inverse proposition being not necessarily true.
65
M. L. SADLER SIMÕES, M. C. T. ABREU VAZ, J. J. R. FRAÚSTO DA SILVA
with the results of the other methods as the value obtained with
4.2.3 — COMPARISON OF METHODS FOR NITRATE
8 minutes. With longer times of distillation the results are still the
DETERMINATION
same.
After studying the chosen analytical methods, comparative studies were carried out between the potentiometric and the steam distillation methods on one
hand, and the spectrophotometric and the steam
distillation methods on the other.
NOTES:
The values given in Table IV arc averages of duplicates. The dry
spinach was obtained by keeping the product in an oven during
48 hours at a temperature of 70°C.
The time of 4 minutes for distallation 130] does not agree as well
For spinach 1) the mean value for the percentage of
nitrogen as nitrate is 0.0251%, the error of the
determination not exceeding 3.5% compared with
average value.
For spinach 2) the mean value is 0.0321% if all the
values are taken into account or 0.0304% if the
value obtained by the potentiometric method using
an aliquot of 20 g is neglected. The value obtained
for the aliquot of 20 g using the potentiometric
method may be a little too high, possibly due to the
interference of chloride ion extracted together with
the nitrate ion.
Table IV
Determination of nitrate in two different samples of spinach by the potentiometric
method and the steam distillation method
N." of
sample "'
Extracting
Analytical
Aliquot of
%o of NO -I :nitrogen
o/o of NOi:nitrogen
solution
method
sample
in Fresh spinach
in dried spinách
As in Ref. 1101
electrode
ca. 20 g
0.0253
0.336
Selective
potent iometry
Steam
distillation
1)
t"
ca. 20 g
As in Ref. 1101
0.0202
0.269
(4 min.)
(4 min.)
0.0242
0.322
(8 min.)
(8 min.)
BaCI,+CdCI,
solution
Idem
0.0258
0.343
(4 and 8 min.)
(4 and 8 min.)
ca. 20 g
0.0355
0.469
10 g
0.0299
0.398
ca.
20 g
Selective
As in Ref. [101
electrode
potentiometry
As in Ref. 1101
BaCI,+CdCI,
2)
solution
Ident
Steam
distillation "
ca.
ca. 20 a
0.0306
0.407
(4 min.)
(4 min.)
0.0320
0.426
(8 min.)
(8 min.)
BaCI,+CdCI ,
solution
Idem
ca. 10 g
0.0285
0.379
(4 min.)
(4 min.)
0.0315
(8 mim)
0.419
(8 min.)
the leaves were lanceolate.
a) Samples I) and 2) correspond to different types of spinach. For sample 2)
distillations
the
rate
of
distillation
was
kept
constant
at
7
ml/min.
according
to the recommended procedure [30].
h) In all
66
Rev. Port. Quim., 21, 61 (1979)
DETERMINATION OF NITRATE AND NITRITE
Indeed the plants generally contain this ion in levels
between 0.5 and 2.0% and chloride has to be lower
than 10 - 'M,otherwise it will interfere with the determination of nitrate ion using specific electrodes.
In the case studied, chloride ion did not interfere
because its concentration in the solution of the
extracted sample was always lower than 10 -1 m.
It therefore appears to be more convenient to use an
aliquot of 10 g of spinach as higher aliquots increase
the probability of having solutions extracted from
spinach with levels of chloride exceeding the values
allowed.
Comparison of the spectrophotometric method,
with the above refered alterations, with the steam
distillation method, has yielded the following
results (Table V):
Table V
Determination of nitrate in two different spinach samples by the spectrophotometric
and the steam distillation method
N." of
Extracting
Analytical
Aliquot
% of NOT-nitrogen
°Jo of NOT-nitrogen
sample" 1
solution
method
sample
in fresh spinach
in dried spinach
ca. 20 g
0.0286
0.380
N1-1 3 /NH 4 CI
Cleve's
1:1
reagent
spectrophotometry
I)
KCI I M
Steam
Distillation
NH,/NH 4 CI
Cleve's
1:1
reagent
ca. 20 g
0.0124
0.166
(4 min.)
(4 min.)
0.0247
(8 min.)
0.328
(8 min.)
ca. 20 g
0.0194
0.258
ca. 20 g
0.0192
0.255
specs ropholometry
2)
BaCI,+CdCl2
BaCI,+CdCl,
Idem
Steam
Distillation
'
ca. 20 g
0.0220
0.293
(4 and 8 min.)
(4 and 8 min.)
a) and b) see Table IV
Two extracting solutions were used here for spinach
2) that indicated by A DRIAANSE and ROBnt RS [38] and
SCHArr and HATCHER', solution [20] for the spectrophotometric method, the values obtained are the
same (within the experimental errors). These values
are identical with the ones obtained by steam distillation.
On the other hand, for spinach I) no influence of
extracting solution can be detected whether it is of
ammonia/ammoniun or of 1 M potassium chloride;
with 8 minutes for distillation the same value is
obtained both for the spectrophotometric method
and the steam distillation method. The value
obtained with 4 minutes for distillation is much
lower and we have noticed that this often happens
when the extracting solution is KCI IM.Using ScHnl 1.
and HATCHER•~ [20] method for sample preparation,
the proteins are precipitated and so, in a general
way, the values obtained with 4 and 8 minutes for
distillation agree within the experimental errors.
Hence the three extracting solutions are all considered to be efficiént and the independent analytical
methods equally accurate.
4.2.4 — DETERMINATION OF NITRITE AND
AMMONIUM
It was not possible to perform the comparative study
of the methods to determine nitrite in spinach since
^-7
M. L. SADLER SIMÕES, M. C. T. ABREU VAZ, J. J. R. FRAÚSTO DA SILVA
in the samples used the nitrite level was always below
The determination of ammonium ion is not within
the, detection limit of steam distillation methods. The
the scope of this work but as it is one of the steps of
spectrophotometric method was therefore the only
the steam distillation method the values are given in
table VI.
one which could he used.
Table VI
Determination of nitrite and ammonium in two
different samples of spinach
N. o
f
sample a)
Extracting
solution
Analytical
method
To of NH 3 -nitrogen
in spinach
07s of NOT-nitrogen
in spinach
Cleve's
reagent
spectrophotometry
1.2x 10 -5
dry
1.6x10
1.2x10
1.6x10
KCI 1 M
Steam
Distillation
<3.5x104
4.6x10-3
BaC1 2 +CdCl 2
Cleve's
reagent
spectrophotometry
3.4xíÕ5
4.5x10-4
BaCl 2 +CdCI,
Steam
fresh
NH 3 /NH 4 CI
1.1
-
5
-
fresh
dry
—
—
2.8x10 - 3
3.7x10-2
1.9x10 3
2.5x10
4
4
I)
2)
3.5x10
4
4.6x10.
3
2
a) See Table. IV.
Note: The values presented for sample I) are averages of duplicates; for sample 2) they are averages of quadruplicates.
Nitrite ion levels in fresh spinach are found to be very low but should ificrease after the harvest.
5 — CONCLUSIONS
thod seems to be accurate in terms of interferences
and detection limit.
This study leads to the conclusion that the best me-
These techniques which we have selected, improved
thod to determine nitrate is the potentiometric one.
and studied in terms of accuracy, precision, sensiti-
In fact, and although its detection limit is higher
vity, detection limit, ease of performance, safety
than of the other methods, it is still good enough for
and practical convenience for routine use, have
the nitrate levels usually found in spinach. On the
been used to determine nitrate and nitrite in some
other hand the recovery percentage of the amount
fresh and refrigerated vegetables.
added to the sample is good (Table II) and so is the
The results obtained will be reported in a following
reproducibility, the best of all methods (Table III).
paper.
As far as accuracy is concerned all methods appear
Received 2. May. 1979
to be equally accurate, since the values obtained for
a given sample are the same for the three independent methods within the experimental errors.
One great advantage of the potentiometric method
relatively to any of the other two is the shorter time
required for its operation, only to be compared with
the spectrophotometric method with autoanalizer,
that needs a more expensive equipment.
ACNOWLEDGEMENT —
To determine nitrite ion and noting its concentra-
tuto Nacional de Investigação Científica (MEC) for financial
tion range in spinach the spectrophotometric me-
support.
68
The authors thank the Insti-
Rev. Port. Quím., 21, 61 (1979)
DETERMINATION OF NITRATE AND NITRITE
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RESUMO
No presente trabalho faz-se uma revisão geral dos métodos
descritos na literatura para a determinação dos ides nitrato
e nitrito em plantas e produtos alimentares. Seleccionados três
métodos que pareceram em princípio, mais convenientes, foram
os mesmos estudados em pormenor em termos de reprodutibilidade, limite de detecção, exactidão, rapidez e conveniência de
execução, apresentando-se alguns resultados da sua aplicação
a amostras de espinafres. Com base nos resultados obtidos
recomenda-se a adopção dos métodos que pareceram mais
adequados para o efeito.
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